A smear below the lowest band could also mean the primers are probably unused and bound together. As Oliver said, there are other possibilities to explore before redesigning the primers. Try reducing the primer concentration and annealing temp, increase template concentration, use fresh dNTPs/polymerase ... Also, note what you get in the region where your amplified product is supposed to be; it will help troubleshoot better and understand which PCR component/step is faulty.

Agree with both Avinash and Oliver...

1. First u must see whether these primers are universal or not. if not u must check self complimentary or loop formation. otherwise try to synthesize new primer pair by aligning different sequences of that region.

2. Try various annealing temperatures.

3. 2-3 different combinations of MgCl2.

4. try to lower amount of dNTP's.

Dear Alexander,

As you mentioned that the primers are designed by yourself, please check the self-compatibility of your primers. In addition, the 3'-end of primers must be compatible with desired template region; if not, DNA polymerase cannot bind to the template-primer hybrid effectively and the reaction would failed. You can check the 3'-end compatibility of primers using NCBI primer BLAST toll.

Be successful

Try annealing temperature from 55 to 60.

Reduce primer concentration.

Check the quality of your templates by using some control primers.

Try to add less MgCl2 as higher Mg is a reason behind less specific amplification

According to your expected band size you may increase annealing time.

try them one by one.

Good Luck

Primer concentration should be reduced. Universal primer for COI is FOLMER Primers (LCO1490 and HC02198) which has annealing temperature of 40.

in addition to above suggestions .... Please keep your primers and reaction mixture at ice cold temperatures and try Hot start PCR.....

i suggest use DMSO for your pcr reaction

good luck

You need to work on correct annealing temperatures. Try gradient PCR may be. Also, while you design primers check the primers properties and design such primers that offers no dimer and secondary structures. There are different softwares available for the assessment of primer properties. Also optimize the primer concentration please.

I hope that will help.

Cheers

Dear Alexander

maybe your primer concentration is high or they are incorrectly designed, So it is better to do as follows:

1. Do a kind of asymmetric PCR (for example add 2mM forward primer to reaction and 1mM reverse ). (this one is very useful)

2.Reduce dNTPs concentration (a little)

3. increase a little Mg concentration.

4. Increase template ( 1-3 Microliter)

5. Try to do PCR at a range of annealing temperature (40-45).

6. After doing this steps, let me look at your gel picture.

I have to solve my problem. The problem was annealing temperature and time. The annealing temperature should be lower than previously. The annealing time should be higher than previosly. Thank you very much for all of your answer. It's really help me indeed.

my friend also conducted research on barcoding Stolus from raja ampat. she use COI F and COI R primer describe by Arn, 1995.  For annealling she use 50  degree celcius. and she succes.